Isolation and characterization of natural products from plant tissue cultures of Maytenus buchananii

Explants of Maytenus buchananii were induced to form a callus aid subsequently to form suspension cultures on a wide variety of media. Culture extracts showed cytotoxic activity, but examination by TLC did not indicate the presence of maytansine. Isolation of natural products from a large scale suspension culture led to the identification of polpunonic acid, sitosterol and the cytotoxic triterpene quinone-methides, tingenone and 22β-hydroxytingenone. Possible biosynthetic relationships of these and other triterpene quinone-methides are discussed.     

A histological study of the Shionogi adenocarcinoma 115 grown in male and female mice

We have previously demonstrated that growth rate and morphology differ between androgen-responsive Shionogi mouse mammary tumours maintained in male and female mice. Furthermore, we can modulate the growth rate of these tumours in male mice by exposing the mice to psychosocial stressors. In the present study, we were interested in determining if tumours in male mice with a comparable growth rate to that in females, also had a morphology similar to that in females. SC115 tumours were examined using histochemical and immunohistochemical techniques. Tumours in male mice were easily distinguishable from tumours in female mice regardless of growth rate. Tumours maintained in female mice contained osteoid-like regions which stained positive for sialic acid and sulphate moieties. No such regions were observed in any of the tumours from male mice. In addition, although all tumours contained MSA (muscle specific actin)-positive and S100 protein-positive cells, these regions were more extensive in the tumours of female mice. This study suggests that tumour growth rate and morphology are independently regulated by the host environment.     

Phenotypic stability of mouse mammary tumor cells cultured on collagen gels

Summary This study demonstrates that phenotypic characteristics of androgen-responsive (AR) Shionogi mouse mammary tumors and androgen-independent (AI) derivatives can be maintained in culture. Cells were seeded onto collagen gels in medium containing 2% dextran-characoal-treated fetal bovine serum with or without 0.01 μg/ml dihydrotestosterone (DHT). Androgen-responsive tumors grew more rapidly than AI tumors in vivo and consequently, cells from AR tumors cultured in DHT-containing medium grew faster than cells in DHT-deprived medium and cells from AI tumors. Androgen-responsive tumors had a sheetlike growth pattern; AI tumors formed clumps or irregular cords of cells. Cells from AR tumors cultured in the presence of DHT formed confluent pavements, whereas cells maintained in the absence of DHT and cells from AI tumors formed clusters or cords of cells. Ultrastructurally, cells of AR tumors were elongated; cells of AI tumors were smaller and rounder. These cellular morphologies persisted in culture. Tumorigenicity of cells was assayed by injecting cells s.c. into host mice. Tumors arising from cells of freshly dissociated AR tumors and cells of AR tumors cultured in the presence of DHT appeared more rapidly and grew faster in intact males than in castrated males and intact females. Tumors arising from cells cultured in the absence of DHT and from freshly dissociated or cultured cells of AI tumors had identical rates of appearance and growth in all hosts. This culture system permits these cells to retain their state of malignant progression in vitro and should be a useful model for studying the origin of heterogeneity within tumors and its role in tumor behavior. This work was supported by a grant from the National Cancer Institute of Canada. J. T. E. is a Research Scholar of the National Cancer Institute of Canada.     

A non‐canonical role of the p97 complex in RIG‐I antiviral signaling

RIG‐I is a well‐studied sensor of viral RNA that plays a key role in innate immunity. p97 regulates a variety of cellular events such as protein quality control, membrane reassembly, DNA repair, and the cell cycle. Here, we report a new role for p97 with Npl4‐Ufd1 as its cofactor in reducing antiviral innate immune responses by facilitating proteasomal degradation of RIG‐I. The p97 complex is able to directly bind both non‐ubiquitinated RIG‐I and the E3 ligase RNF125, promoting K48‐linked ubiquitination of RIG‐I at residue K181. Viral infection significantly strengthens the interaction between RIG‐I and the p97 complex by a conformational change of RIG‐I that exposes the CARDs and through K63‐linked ubiquitination of these CARDs. Disruption of the p97 complex enhances RIG‐I antiviral signaling. Consistently, administration of compounds targeting p97 ATPase activity was shown to inhibit viral replication and protect mice from vesicular stomatitis virus (VSV) infection. Overall, our study uncovered a previously unrecognized role for the p97 complex in protein ubiquitination and revealed the p97 complex as a potential drug target in antiviral therapy.     

Creation of an in vitro biomechanical model of the trachea using rapid prototyping

Previous in vitro models of the airways are either rigid or, if flexible, have not matched in vivo compliance characteristics. Rapid prototyping provides a quickly evolving approach that can be used to directly produce in vitro airway models using either rigid or flexible polymers. The objective of this study was to use rapid prototyping to directly produce a flexible hollow model that matches the biomechanical compliance of the trachea. The airway model consisted of a previously developed characteristic mouth–throat region, the trachea, and a portion of the main bronchi. Compliance of the tracheal region was known from a previous in vivo imaging study that reported cross-sectional areas over a range of internal pressures. The compliance of the tracheal region was matched to the in vivo data for a specific flexible resin by iteratively selecting the thicknesses and other dimensions of tracheal wall components. Seven iterative models were produced and illustrated highly non-linear expansion consisting of initial rapid size increase, a transition region, and continued slower size increase as pressure was increased. Thickness of the esophageal interface membrane and initial trachea indention were identified as key parameters with the final model correctly predicting all phases of expansion within a value of 5% of the in vivo data. Applications of the current biomechanical model are related to endotracheal intubation and include determination of effective mucus suctioning and evaluation of cuff sealing with respect to gases and secretions.     

A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation.